The broad goal of the proposed research is to gain a deeper understanding of the molecular mechanisms underlying hemophilia B. The blood coagulation cascade is a complex biochemical system that is regulated extensively in order to achieve hemostasis without inducing thrombosis. A key component in the activation and regulation of sustained coagulation is a protein complex known as the intrinsic Xase. This complex consists of the Factor IXa (fIXa) enzyme, along with its allosteric activator Factor VIIIa (fVIIIa) and several metal ions which also modulate its activity. Hemophilia B is caused by dysfunction of fIXa, the catalytic subunit of the intrinsic Xase. A specific subset of mutations tht cause hemophilia B do not affect the concentration of fIXa in the blood and do not significantly destabilize the protein. However, these mutations do impair the catalytic function of fIXa. We hypothesize that these mutations perturb conformational equilibria that are critical for allosteric communication, substrate binding, and enzymatic turnover. In order to test this hypothesis, we will first dissect allosteric communication pathways in fIXa using multi-temperature X-ray crystallography and computational approaches. These analyses will identify correlated conformational heterogeneity and energetic coupling between distal regions of the enzyme. Next, we will apply this same methodology to fIXa point mutants that are associated with hemophilia B. Combined with functional assays, this information will allow us to quantitatively understand how mutational perturbations to the conformational ensemble effect enzymatic activity. Finally, we will rationally engineer fIXa mutations whose effects suppress the disease phenotype in hemophilia B variants. This strategy of rescuing function by restoring the native fIXa conformational ensemble will inform future development of therapeutics by identifying allosteric networks that can be targeted with small molecules. Looking forward, the methodology of biophysical rescue that we will establish can be applied to other genetic diseases that result from single point mutations.